Prior art concerning the propagation of the Vitis species by tissue culture methods is not that prevalent. Currently, there is one report which deals with vine production from fragmented shoot apices; M. Barlass and K. G. M. Skeen (1978), "In vitro propagation of grapevine (Vitis vinifera L.) from fragmented shoot apices." Vitis 17: 335-340. There are two reports which demonstrate the feasibility of producing vines via somatic embryogenesis, Krul and Worley, "Formation of Adventitious Embryo in Callus Cultures of `Seyval`", J. Amer. Soc. Hort. Sci. 102: 360-363; and M. G. Mullins et al (1976), Somatic Embryos and Plantlets from an Ancient Clone of the Grapevine (cv. Cabernet-Sauvignon) by apomixis in vitro. J. Expt. Bot. 27: 1022-1030. It is possible that grape propagation by shoot tip multiplication may require little or no modification of established methods and therefore, is not reported. See T. Murashige (1974), Plant Propagation Through Tissue Cultures, Annu. Rev. Plant Physiol. 25: 135-166.
In plant propagation, by somatic embryogenesis, i.e. the production of embryonic structures (identical to those in seeds) from isolated plant cells was described in the late 1950's by F. C. Steward et al (1958), Growth and Organized Development of Cultured Cells; II. Organization in cultures grown from freely suspended cells. Amer. J. Bot. 45: 705-708; and J. Reinert, Uber die Kontrolle der orphogenese und die Induktion von Adventivembryonen an Gewebekulturen aus Karotten, Planta 53: 318-333.
Embryoids of grape such as other plants arise from proembryonic masses embedded in callus cells. The factors involved in the initiation of proembryonic masses are not known. The transformation of the proembryonic masses to embryoids in grape occurs when they are shifted from a medium containing a active auxin to one containing a less active one; or when the cytokinin level of the medium is lowered. A highly embryogenic callus of `Seyval` has been isolated which does not require growth regulators for growth. Embryoid formation in this callus occurs when it is grown on auxin for a short while and then cultured on auxin-free media, when the calcium concentration is one half that of the Murashige-Skoog formulation, or when the cultures are grown for long intervals without subculture. Most of the vines regenerated from the original `Seyval` callus have appeared to be normal. However, a subpopulation of these embryos did not develop normally but produced secondary somatic grape embryos at the junction of the root and shoot.
The development of normal vines from these etiolated secondary grape embryoids is complicated by separate dormancies of the roots, hypocotol, cotyledons and shoot apices, each of which respond to different physical and chemical stimuli.
The processes of successful embryoid production and normal vine development have appeared to be mutually antagonistic. For instance, embryoids which produce embryoids generally senesce and die shortly after embryoid formation. In contrast, embryoids which develop flattened green separate cotyledons or normal shoots generally lose their capacity for the production of secondary embryoids. Therefore, treatments which encourage embryoid production do not favor normal vine development, and conversely, treatments which favor normal vine development restrict somatic embryogenesis.
The production of vines or other plants from isolated cells via somatic embryogenesis is essential for progress in anticipated genetic modifications for plant improvement. Such strategies may involve manipulation in selections of cells tolerant to physical stress (cold or heat) or chemical stress, (herbicidal, bacterial and fungal toxins) or the uptake of gene vectors by protoplasts from embryogenic cells. The use of somatic embryogenesis as an alternative method for the massive production of plants in particular vines has possibilities, but heretofore methods for most cultivars have yet to be developed.
The basic procedures for the mass clonal propagation of certain plants via epidermal cells of somatic embryos is set forth in a publication "In Vitro Propagation of Grape", W. R. Krul and Myerson (1980); Journal Article Number 1932 Rhode Island Agricultural Experimental Station. Also, a detailed discussion of this field is found in a thesis entitled "Adventitious Embryo-genesis and Plantlet Development in Cultures of Vitis vinifera L. `Seyval`", Judith Myerson thesis submitted to the University of Rhode Island presently on file at the Library of the University of Rhode Island, Kingston, R.I.
I have discovered a method and the plants produced thereby in which somatic embryos may assume either a self-replicating mode or a vine development mode. Prior to my present invention, when a process was employed which was successful in producing self-replicating embryos, the development of normal plants from such embryos occurred at a frequency of about 5%. The present invention permits development of functional plants from such embryos at a frequency in excess of 90%. With the present invention, it is possible to maintain a number of plants in the replicative mode and induce functional plants at times convenient for a producton schedule. The invention is exemplified through the clonal propagation of grape via epidermal cells of somatic embryos of the Vitis cultivar specifically the hybrid `Seyval`.
Broadly, the cloning process includes removing somatic embryos from a `mother` plant (see the Drawing, cycle A). Such embryos are cultured for a first time period on a medium, (M/S medium without growth regulators). In this medium, the embryos self-replicate. Subsequently, those embryos which are selected for vine development (see the Drawing, cycle B) are transferred to the same medium with a growth regulator having cytokinin activity and maintained for a fixed period of time in the medium until the desired level of embryo development is reached. At this stage, the embryos are transferred to the same medium without the growth regulator and allowed to develop to allow transplant to soil.